Glucagon is very useful in type 1 diabetes, both as a rescue drug given in large doses to persons who have severe hypoglycemia, and in small doses in the setting of automatic closed loop glycemic management. In solution, glucagon is highly unstable, and must be reconstituted often for the closed loop application.
In the 1950's, after its purification and crystallization, glucagon was also found to form many slender fibrillar structures in solution (Staub A and Behrens O K, J Clin Invest 33, 1629-1633 (1954), incorporated by reference herein). This fibrillation increases with incubation time; further aging of glucagon solutions leads to gels with high concentrations of packed fibrils. The fibrils have a beta-pleated sheet amyloid protein configuration. Though some compounds, including cyclodextrins, have been reported to reduce the tendency to form these fibrils (Matilainen L et al, J Pharm Sci 97, 2720-2729 (2007 and Matilainen L et al, Eur J Pharm Sci 36, 412-420 (2008) both of which are incorporated by reference herein) there is no additive or method that is known to block their formation. In 2004, fibrillated glucagon was reported to be cytotoxic in cultured mammalian cells (Onoue S et al, Pharm Res 21, 1274-1283 (2004), incorporated by reference herein). The authors emphasized the potentially dangerous nature of these fibrils and pointed out the presence of similar fibrils in Alzheimer's-, Parkinson's- and prion diseases.
First mentioned in 1964 (Kadish A, Am J Med Electron 3, 82-86 (1964) incorporated by reference herein,) there has been a resurgence of interest in the automated delivery of glucagon during frequent glucose measurement in order to prevent hypoglycemia in people with type 1 diabetes. In the closed loop setting, several reports showed benefit of glucagon administration in animals (El-Khatib F H et al, J Diabetes Sci Technol 3, 789-803 (2009) and Ward W et al, IEEE Sensors Journal 8, 88-96 (2008), both of which are incorporated by reference herein) and in humans (Castle J R et al, Diabetes Care 33, 1282-1287 (2010) and El-Khatib F G et al Sci Transl Med 2, 27ra27 (2010), both of which are incorporated by reference herein). In the Castle (2010) reference supra, the duration of time in the hypoglycemic range for a bihormonal approach including both glucagon and insulin was less than half the duration for the insulin-only experiments. The doses of subcutaneous glucagon in closed loop studies are generally small, and range from 30-180 μg. To perform these studies, the glucagon must be frequently reconstituted to minimize fibril formation. Frequent reconstitution is not a viable option for treating patients.
Glucagon is readily soluble in alkaline conditions (around pH 10) and at 37° C., there is much less amyloid fibril formation in glucagon solubilized under alkaline conditions than compared to glucagon solubilized under the acidic conditions (about pH 3) commonly used in currently available commercial preparations of glucagon (Ward W K et al, J Diabetes Sci Technol 4, 1311-1321 (2010), incorporated by reference herein). Glucagon aged for 5 days at a pH of 8.5 showed some cytotoxicity at pH 8.5 at a concentration of 2.5 mg/ml. Glucagon aged at pH 10 in glycine, showed no cytotoxicity. However, glucagon solubilized at alkaline conditions spontaneously degrades.
Therefore, what is clearly needed is a stable glucagon formulation that inhibits fibril formation and resists glucagon degradation for at least three days and preferably seven days so that the glucagon formulation may be administered in a pump.
Curcumin reduces Aβ fibril formation in vitro and when administered systemically to transgenic rodents predisposed to Alzheimer's disease (Hamaguchi T et al, Am J Pathol 175, 2557-2565 (2009) incorporated by reference herein). Curcumin has also been reported to inhibit the polymerization of α-synuclein, the compound responsible for Lewy body formation in diseases such as Parkinson's disease (Ono K and Yamada M, Curr Pharm Des 14, 3247-3266 (2008) and Ono K et al Curr Pharm Des 14, 3247-3266 (2008); both of which are incorporated by reference herein). This finding was confirmed in Pandey N et al, Acta Neuropathol 115, 479-489 (2008); which is incorporated by reference herein. However, curcumin stored in a glycine buffer at pH 9.0 degrades quickly with a half-life of about 7 hours. While this half-life could be extended to 12.5 hours in a glucagon buffer and further to 33.5 hours with the further addition of human serum albumin, this is not a sufficiently stable for the purposes of an automated delivery system.